Bildgebende Massenspektrometrie Erfolgreiche Anwendungen in der biomedizinischen Forschung Markus Stoeckli
MALDI Mass Spectrometric Imaging Principle 2 MALDI MSI Markus Stoeckli September 2014
Comparison of Human Lung Tumor Blots Squamous Cell Carcinoma Squamous Cell Carcinoma Adenocarcinoma 2000 4000 6000 8000 10000 12000 14000 16000 Mass (m/z) 3 MALDI MSI Markus Stoeckli September 2014
Peptides on Mouse Brain Tissue 4 MALDI MSI Markus Stoeckli September 2014
What s wrong with this slide? 5 MALDI MSI Markus Stoeckli September 2014
The Vision in 2000 6 MALDI MSI Markus Stoeckli September 2014
Drug Discovery Process Source: http://www.novartis.com/innovation/research-development/drug-discovery-development-process/index.shtml 7 MALDI MSI Markus Stoeckli September 2014
Amyloid Beta Peptides Aß-(1-40) Aß-(1-42) 8 MALDI MSI Markus Stoeckli September 2014 stained
Cyclic Peptide Intra Muscular Deposit, 1 week post dose 9 MALDI MSI Markus Stoeckli September 2014
Proteins and MS Imaging some difficulties Membrane Proteins Source: Ghaemmaghami, Nature (2003), 425, 737-741 10 MALDI MSI Markus Stoeckli September 2014
More about Proteins Separation is key High concentration range Low MS sensitivity in high mass range Suppression effect during ionization Targeted Px Extraction, digest, fractionation, separation, SRM Source: Picotti, Nat. Methods, 2008, 5 (11), 913-4 11 MALDI MSI Markus Stoeckli September 2014
MSI on Proteins A Reality Check Data from IMSC 2014 MS Imaging On-tissue digest, MALDI TOF 250 peptides, 109 proteins (Bruker, Arndt Asperger) LC/MS DIA, variable width 20 k peptides, 2000 proteins (Ying Zhang) HILIC, fractionations: 7000 proteins (Nathalie Selevsek) 12 MALDI MSI Markus Stoeckli September 2014
Small Molecules and MSI Background LCMS dissection of animal limited spatial resolution only parts of animal WBAL label required total radioactivity not specific 13 MALDI MSI Markus Stoeckli September 2014
MALDI MS Imaging Platform Current setup at NIBR Animal Dosing Sectioning Matrix Coating Data Acquisition Data Cube Generation Data Processing and Visualization 14 MALDI MSI Markus Stoeckli September 2014
Compound Distribution 24 h post dose, MRM 15 MALDI MSI Markus Stoeckli September 2014
Hydroxylated Compound Distribution 24 h post dose, MRM 16 MALDI MSI Markus Stoeckli September 2014
LLOD/LLOQ Signal 10000 1000 R² = 0.9989 100 10 1 0.1 0.01 0.1 1 10 100 1000 Tissue Concentration [um] 17 MALDI MSI Markus Stoeckli September 2014
MS Imaging Workflow Compound and metabolite distributions Request from Team MS Method Development LLOD on Liver Dosing Matrix Deposition Dehydration & Formatting Transfer Embedding & Sectioning Data Acquisition Data Processing Reporting 18 MALDI MSI Markus Stoeckli September 2014
Workflow and Timelines Typical times, depending on experimental design 1 week Identification of compound & external standard Request background info (PK data) and compound LLOD of compound on plate and tissue, MS/MS method (2 d) Write study protocol (1/2 d) Dosing (data, organization, treatment, sacrifice, freezing, sectioning, 5 to 8 d) Measurement of sections (5 to 10 d) Data evaluation (5 to 15 d) 19 MALDI MSI Markus Stoeckli September 2014
Automation 20 MALDI MSI Markus Stoeckli September 2014
Matrix Deposition Artifacts 21 MALDI MSI Markus Stoeckli September 2014
imatrixspray Free and open source software and hardware Fully automated Multiple reagents Optimized for MALDI MSI Automation friendly imatrixspray.com 22 MALDI MSI Markus Stoeckli September 2014
High Mass Resolution and Specificity OrbiTrap, 100 000 resolution FlashQuant, SRM 23 MALDI MSI Markus Stoeckli September 2014
Time Series 8 h M1 (533 > 446) Cpd (535 > 448) M2 (551 > 449) M3 (551 > 464) M4 (727 > 551) Optical 24 MALDI MSI Markus Stoeckli September 2014
Time Series 24 h 25 MALDI MSI Markus Stoeckli September 2014
Time Series 48 h Optical 26 MALDI MSI Markus Stoeckli September 2014
Time Series 72 h Optical 27 MALDI MSI Markus Stoeckli September 2014
Quantification Methods Internal Standard 4. MALDI Matrix 3. Internal Standard (Isotope or similar) 2. External Standard or Spiked Tissue 1. Tissue Section of Dosed Animal c I I Cpd IntStd I I IntStd ExtStd c ExtStd 28 MALDI MSI Markus Stoeckli September 2014
Data Evaluation and Reporting 3h, 10 mg/kg, 3 h p.o., 3 separate sections from same animal 1400.0 1200.0 c (i) [µm] 1000.0 800.0 600.0 400.0 200.0 0.0 29 MALDI MSI Markus Stoeckli September 2014
Direct Comparison QWBA/MSI BT-474 tumor-bearing nude mice 10 mg/kg [ 14 C]-labeled compound 20 15 Brain [um] 15 Lung 10 10 5 5 0 0.5h 2h 4h 8h 0 0.5h 2h 4h 8h [um] 50 Liver [um] 40 Tumor 40 30 20 10 30 20 10 0 0.5h 2h 4h 8h 0 0.5h 2h 4h 8h 30 MALDI MSI Markus Stoeckli September 2014
WBAL Compared to MALDI 10 mg/kg p.o., 2 h post dose WBAL Kidney Tumor Lung Brain Compound Concentration Bile Liver compound / desmethyl 2 8 24 31 MALDI MSI Markus Stoeckli September 2014
Alternatives to MALDI MSI Sensitivity is key Source: M.-Z. Huang et al. / Analytica Chimica Acta 702 (2011) 1 15 32 MALDI MSI Markus Stoeckli September 2014
Surface Sampling Tissue concentrations by liquid extraction from tissue surface Compound 30000 25000 Metabolite 20000 15000 10000 5000 MSi LESA 0 blood brain liver stomach content 33 MALDI MSI Markus Stoeckli September 2014
WBAL Brain Penetration 50 um spatial resolution, nm sensitivity Source: Esther von Kerkhof, Novartis 34 MALDI MSI Markus Stoeckli September 2014
PET/CT of Biologics Quantification, Resolution - PK, Biodistribution 124 I-labeled Ab fragment 64 Cu-DOTA-labeled Ab frag. 35 MALDI MSI Markus Stoeckli September 2014
Lung Sections of TB-Infected Rabbits 30 µm Pixel Imaging of PA824 in Granuloma 36 MALDI MSI Markus Stoeckli September 2014 Brendan Prideaux
PE ** Compound Optical Scalp Biopsy Sections measure by SIMS High resolution Control PG/OL CSF Nanoparticles PG 3 13 7 4 5 0 0 0 0 0 39 43 43 36 0 52 0 0 0 0 0 37 MALDI MSI Markus Stoeckli September 2014
http://maldi.ms A starting point for MS imaging 38 MALDI MSI Markus Stoeckli September 2014
Acknowledgment Prakash Mistry Fabian Von Arx Fabienne Schaeffer Robert Cozens Francesco Hofmann Masato Murakami Dieter Staab Brendan Prideaux Nicole Ehrhard Gregory Morandi Rocco Falchetto Steve Martin ABSciex 39 MALDI MSI Markus Stoeckli September 2014
40 MALDI MSI Markus Stoeckli September 2014